PROJECT SUMMARY/ABSTRACT Nucleotide excision repair (NER) is the primary method to repair DNA adducts formed by platinum (Pt)-based chemotherapies, such as cisplatin. Stratifying cancer patients based on mutations in NER genes that disrupt NER activity and sensitize cells to Pt-based chemotherapeutics represents a promising precision medicine strategy. To address this opportunity, the long-term objectives of this proposal are (i) to develop a robust pipeline that can derive validated, phenotypic insights from patient genomic data, and (ii) benchmark this pipeline on the essential NER scaffold protein, Xeroderma Pigmentosum Complementation Group A (XPA). A computational approach will be developed to predict missense mutations in XPA that disrupt NER activity by integrating protein stability modeling, proximity to known disease-causing mutations, secondary structure prediction, and evolutionary conservation. Predicted deleterious mutations in XPA will be functionally validated by in vitro and cell-based NER activity assays, as well as by cell survival assays after treatment with cisplatin. The mechanism of NER dysfunction and cisplatin sensitivity for a subset of validated mutations in XPA will be determined using multiple biophysical and structural approaches, including analyses of DNA binding affinity, secondary structure and stability, three-dimensional structure, and interactions with NER pre-incision complex proteins. Completion of this proposal will provide mechanistic insights into how a subset of missense mutations in XPA from tumor genomic data disrupt NER activity and ultimately sensitize cells to cisplatin. Accurately selecting good- versus poor-responders to Pt-based chemotherapy treatment has potential to improve patient quality of life and overall survival, allow for earlier implementation of alternative strategies for poor-responders, and decrease unnecessary costs of care. In addition, this proposal will lay the foundation for a robust strategy capable of deriving mechanistic insights into cell phenotype from genomic data, that can be extended to all core NER pathway proteins or applied to other clinically-relevant pathways. Importantly, completion of this proposal will equip the applicant with a uniquely versatile, multidisciplinary skillset that will greatly enhance the applicant?s potential and career as a future independent investigator. Support from the selected institution and Sponsor, regular progress meetings with the co-sponsoring team and collaborators, strong multi-tiered mentoring, and participation at national conferences will ensure the applicant receives a well-rounded training experience for the duration of this fellowship.